Cryo-Electron Tomography-Guided Nanoscale Epitope Mapping for Quantitative Assessment of PD-L1 Conformational Dynamics in Triple-Negative Breast Cancer Microenvironments

Abstract

Triple-negative breast cancer (TNBC) poses significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME) and limited actionable targets. Conventional immunohistochemical PD-L1 quantification fails to resolve spatiotemporal conformational heterogeneity critical for checkpoint inhibitor responsiveness. Here, we present a cryo-electron tomography (cryo-ET)-integrated epitope mapping framework coupled with sub-tomogram averaging and molecular dynamics (MD) simulation to characterize PD-L1 conformational states at nanometer resolution across patient-derived TNBC xenograft specimens. Applying this pipeline to 47 biopsy-matched cohorts, we identified three distinct PD-L1 conformational populations correlated with differential CD8⁺ T-cell infiltration density (p < 0.001). Furthermore, allosteric modulation indices derived from MD trajectories predicted atezolizumab binding affinity with 89.4% accuracy. These findings establish a quantitative structural framework for precision immunotherapy stratification in TNBC.